Autophagy and endocytosis are essential in regulating cellular homeostasis and cancer immunotherapeutic responses. Existing methods for autophagy and endocytosis imaging are susceptible to cellular micro-environmental changes, and direct fluorogenic visualization of their fluxes remains challenging. We develop a novel strategy via clicking of organelle-enriched probes (COP), which comprises a pair of trans-cyclooctenol (TCO) and tetrazine probes separately enriched in lysosomes and mitochondria (in autophagy) or plasma membrane (in endocytosis). These paired probes are merged and boost a fluorogenic click reaction in response to autophagic or endocytic flux that ultimately fuses mitochondria or plasma membrane into lysosomes. We demonstrate that this strategy enables direct visualization of autophagic and endocytic fluxes, and confer insight into correlation of autophagic or endocytic flux to cell surface expression of immunotherapeutic targets such as MHC-I and PD-L1. The COP strategy provides a new paradigm for imaging autophagic and endocytic fluxes, and affords potential for improved cancer immunotherapy using autophagy or endocytosis inhibitors.
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